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Extranodal oral lymphoma. Part I. A morphologic and immunoperoxidase study of 34 cases
Extranodal oral lymphoma. Part I. A morphologic and immunoperoxidase study of 34 cases Janice P. Handlers, D.D.S., * Robin E. Howell, D.D.S., ** Albert M. Abrams, D.D.S., MS., * and Raymond J. Melrose, and Halifax, Nova Scotia, Canada UNIVERSITY DALHOUSIE
OF SOUTHERN UNIVERSITY
CALIFORNIA
SCHOOL
OF DENTISTRY
AND
D.D.S., * Los Angeles, Cal&
FACULTY
OF DENTISTRY.
Thirty-four cases of oral lymphoma were classified by the Lukes-Collins system on the basis of morphology and immunoperoxidase staining. Ninety-seven percent of these were morphologically identified as B-cell neoplasms: 6% SCFCC, 9% LCFCC, 26% SNCFCC, 24% LNCFCC, 12% IBS, and 16% malignant plasma cell proliferations. Monoclonal immunoperoxidase staining for cytoplasmic immunoglobulin was positive in 41% of the cases overall, but 100% of the cases of immunoblastic sarcoma and malignant plasma cell lesions stained positively. (ORAL SURC. ORAL MED. ORAL PATHOL. 61:362-367, 19861
T he use of the immunoperoxidase nique as an aid in the diagnosis
staining techof lymphomas occurring within lymph nodes has been well established in the literature.‘** There are few reports, however, of its use in extranodal disease, particularly that found in the head and neck area. Matthews and Basu reported a series of eleven cases of nonHodgkin’s lymphoma’ and eight cases of plasma cell lesions4 arising within the oral cavity in which this technique was used. Eight of the cases of nonHodgkin’s lymphoma and five of the plasma cell lesions stained positively for monoclonal immunoglobulin. Wright and associate9 examined four oral plasma cell lesions by immunoperoxidase methods, and all of them showed monoclonal staining. These studies suggest that the immunoperoxidase method is valuable in differentiating neoplastic lymphoid processes from inflammatory conditions and from nonlymphoid neoplasms on the basis of monoclonal This
project
was
funded
by
University
School of Dentistry Biomedical 2S07RR05303-21 and the Department University. *Department of Pathology. University School of Dentistry. **Division Faculty
362
of Oral of Dentistry,
Pathology, Dalhousie
of Southern Research of Oral
Department University.
of
California
Support Grant Biology, Dalhousie Southern of
California Oral
Biology,
staining. In addition, the immunoperoxidase technique is of value in determining the lineage of the neoplastic population, particularly when correlated with the morphologic appearance of the cell.6 It has been shown that approximately 50% of B-cell lymphomas will retain antigenicity of immunoglobulin markers after routine formalin fixation and processing.’ On the basis of a large number of cases studied by multiple morphologic and immunologic parameters, Lukes and Collins have outlined morphologic characteristics which are even more reliable than immunohistochemical stains of processed tissue in differentiating B-cell and T-cell lymphomas.7~8 The characteristics of B-ceil lymphomas are summarized in Table I. The prognostic significance of the Lukes and Collins classification of lymphomas is still somewhat controversial. However, it appears that immunoblastic sarcomas and noncleaved follicular center-cell (transformed) lymphomas have a poorer prognosis.9 It is well documented that there is a significant difference in the prognosis for nodular and diffuse lymphomas as classified by Rappaport.” In nodal lymphomas, the nodular lymphomas appear to have a distinctly better survival rate than those with a diffuse pattern of infiltration.gx “2 ‘* In this study, forty-one cases of potentially malig-
Volume Number
Extranodal
61 4 TABLE
1:
LUKES-COLLINS
CLASSIFICATION
OF
B-CELL
NU&W
Cyt0plasmic
Characteristics
Characteristics
Characteristics
1. Small Lymphocytic Lymphoma
Small cell 6 - 9 urn Uniform
Clumped chromati” Round, distinct border Rare mitoses
scanty Indistinct
2.
Small Cleaved Follicular Center Ceil (SCFCC)
Small cell 6. 12 urn Slight variation
Marked Irregularities Indented, Lobulated, Cleaved Clumped, dense chromatin Few-rare mitoses
Indistinct
3.
Large Cleaved Follicular Center Cell ILCFCC)
Large cell 20 40 urn Marked variation in size and shape
Coarse chromatin clumping Vesicular nuclei Marked irregularities Few-rare mitoses
Indistinct
4.
Small Non-Cleaved Follicular Center Cell (SNCFCC)
Medium
Multiple distinct nucleoli Distinct nuclear borders Rounded Numerous mitoses
Indistinct
5.
Large Non-Cleaved Follicular Center Cell (LNCFCC)
Large cell 20 - 40 urn
Vesicular Small nucleoli on nuclear membrane Irregularity in size & shape Numerous mitoses
I “creased Deep staining Lacks plasmacytoir’ features
6.
lmmunoblastic Sarcoma (16s)
Large cell 20
One or mcwe prominent nucleoli Much variation in size Dense chromatin clumping on nuclear membrane Numerous mitoses
Moderate to abundant Eosinophilic Distinct membrane Marked pyroninophilia Plasmacytoid
7.
Plasmacytoid Lvmphocytic Lymphoma
Small cell 6
Small, round Uniform Plasmacytoid Few to no mitoses
Distinct Eosinophilic Eccentric nucleus Plahmacytoid
6.
Plasmacytoma/ Multiple Myeloma
Ovoid cell Large cell > 20 urn
Distinct membrane Rounded Clockface (Peripheral) chromatin clumping Few mitoses
Abundant Eorinophilic Pyrinophilic Distinct membrane Eccentric nucleus
20 urn
40 urn
10 urn
nant lymphoid lesions were evaluated by routine stains and by immunohistochemical means to determine the number of routinely fixed and processed oral cases in which the lymphocytic infiltrate stained in a monoclonal manner by the immunoperoxidase technique using polyclonal test sera. Two of these were eliminated from the study because they were interpreted to be Hodgkin’s lymphoma, and three were eliminated because they were interpreted and confirmed by other studies to be leukemic infiltrates. The tumors were also classified according to the method of Lukes and Collins in order to determine what percentages of these oral lymphomas were composed of the different cell types described in this classification system (B-cell, T-cell, histiocytic, etc.). In a subsequent article the results of the morphologic and immunoperoxidase study of oral lymphomas will be correlated with clinical features and prognosis.
METHODS
363
LYMPHOMAS
C.dlldJ~
cell 15
oral lymphoma
AND MATERIALS
All cases with diagnoses of malignant lymphoma, lymphoproliferative process, plasmacytoma, multiple myeloma, or malignant tumor suggestive of lymphoma or plasmacytoma, received between January 1974 and January 1984, were selected from the files of the University of Southern California Oral Pathology Laboratory. Clinically, these lesions were all extranodal, located within intraoral soft tissue, mandible, or maxilla. Paraffin-embedded tissue was available in forty-one cases. Hematoxylin and eosin, methyl green pyronine, periodic acid-Schiff, and Giemsa stains were performed in each case. Specimens were also stained by the immunoperoxidase technique for IgG (1:500), IgM (1:300), IgA (1:300) and IgD (most cases) (1:300), kappa (l:lOOO), lambda (l:lOOO), and muramidase (1:300) as described previously. l3 All materials were obtained
364
0”
Handlers et al.
SCFCC
.
LCFCC
SNCFCC
LNCFCC
Oral April,
IBS
P,M
Surg. 1986
Lym#locytic
1. Total numberof caseswithin eachLukes-Collins category plotted in order of advancingB-cell transformation.
Fig.
from DAK0 of Santa Barbara, California, and Hornby, Ontario, with the exception of the rabbit PAP reagent, which was obtained from Zymed of Hornby, Ontario. Briefly, paraffin sections were deparaffinized and rehydrated. They were then placed in a hydrogen peroxide-methanol bath for 30 minutes at room temperature to block endogenous peroxidase activity. After a 5-minute bath in Tris buffer, pH 7.6, they were treated with the appropriate rabbit anti-human antisera and incubated for 30 minutes. Another 5-minute Tris wash was followed by application of swine anti-rabbit immunoglobulin, a 20-minute incubation, and a 5-minute wash. Rabbit PAP reagent was applied and incubated for 20 minutes. A Tris buffer wash preceded application of the chromagen diaminobenzidine prior to incubation of the tissue for 5 minutes. The slides were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted with Permount. Tonsillar tissue and inflamed gingival tissue were used for positive controls. Negative controls were achieved by omitting the primary antibody and substituting normal swine serum. All sections were then studied and classified by the Lukes and Collins system of classification as
described for nodal lymphomas.7*8 The immunoperoxidase sections were evaluated for positivity against the respective controls. These results are tabulated in Table II. RESULTS
Of the forty-one cases, five were eliminated from the study because they were interpreted morphologically to represent Hodgkin’s disease (two cases) or myelomonocytic leukemic infiltrates (three cases). Two of the cases showed polyclonal staining for IgG, IgA, IgM, kappa, lambda, and muramidase. These
Fig. 2. A, Malignant plasmacell tumor showingnegative immunoperoxidase staining for lambda light chains. (Magnification, x250.) B, Samecasedemonstratingpositive monoclonalintracytoplasmicstaining for kappa light chains. This is demonstratedby the presenceof dark granular material within the cytoplasm,in contrast to the clear cytoplasmseenin A. (Magnification, x250.)
were interpreted on this basis, as well as morphologically, to represent inflammatory lesions and were not considered further. Fourteen of the remaining thirty-four cases (41%) showed monoclonal cytoplasmic staining. Classification of the tumors by the Lukes and Collins classification revealed that 6% of the thirtyfour cases were small-cleaved follicular center cell (SCFCC) lymphomas, 9% were composed of largecleaved follicular center cells (LCFCC), 26% of small noncleaved (small transformed) follicular center cells (SNCFCC), and 24% of large noncleaved (large transformed) follicular center cells (LNCFCC). Immunoblastic sarcoma (IBS) accounted for 12% of the cases, malignant plasma cell proliferations (plasmacytoma or multiple myeloma) for 18%, and plasmacytoid lymphocytic lymphoma for 3%. One case (3%) of true (muramidase-positive) histiocytic lymphoma was found (Fig. 1). None of the small-cleaved or large-cleaved follicu-
Volume Number
Extranodal
61 4
oral lymphoma
365
% loo-
Fig. 3. Immunoblastic sarcoma stained with periodic acid-Schiff stain.The cellsshowprominentnucleoli,dense chromatin clumping on the nuclear membrane,moderate to abundant cytoplasm with a distinct membrane,and numerousmitoses.(Magnification, x250.)
lar center cell lymphomas stained for immunoglobulin or muramidase. Eleven percent (one of nine) of the small noncleaved follicular center cell lymphomas stained positively. This case was positive for IgM only and was one of two cases of SNCFCC lymphoma of the Burkitt type in our series. Interestingly, although the other case was negative in our series, a repeat biopsy studied at another laboratory was positive for IgM and kappa chain. One of eight (13%) of the large noncleaved follicular center cell lymphomas stained positively (K only). In contrast to the above-mentioned tumors, all tumors (100%) classified as plasmacytoma/multiple myeloma (six of six) (Fig. 2), immunoblastic sarcoma (four of four) (Fig. 3), and plasmacytoid lymphocytic lymphoma (one of one) stained positively (Fig. 4). DISCUSSION
Large multiparameter studies of nodal lymphomas have shown that B-cell neoplasms account for the majority of non-Hodgkin’s lymphoma.9, 14-16Studies of extranodal oral lymphomas, although few, also appear to support this contention. In a morphologic study of eight cases of non-Hodgkin’s lymphoma of the hard palate by Blok and associates,17 seven were interpreted as B-cell neoplasms. Hashimoto and Kurihara** interpreted all of their nine cases of oral lymphoma as B-cell neoplasms on the basis of morphologic appearance. The results of our study were consistent with these findings. On the basis of morphologic examination, all but one of the thirtyfour cases were classified as B-cell lymphomas. The one exception was classified as a “true” histiocytic
0
I SCFCC -B-CELL
1 1 SNCFCC 1 LCFCC LNCFCC TR*NSFORM*TION
I ISS
I PIM
1 Plaamacytoid Lymphocytic m
Fig. 4. Percentageof tumorswithin eachLukes-Collins category showingpositive monoclonalstaining for cytoplasmic immunoglobulinplotted in order of advancing B-cell transformation.
lymphoma, and this was supported by positive immunoperoxidase stains for muramidase. Of the remaining thirty-three cases with B-cell morphology, thirteen (39%) were confirmed on the basis of monoclonal immunoperoxidase stains for cytoplasmic immunoglobulin to represent B-cell neoplasms. Other similar studies have reported a higher percentage of lymphomas showing positive staining for immunoglobulins. Matthews and Basu,3 in a study of eleven oral lymphomas, reported positive staining in 64%. In studies of nodal lymphomas, Taylor’ has reported positive monoclonal patterns in approximately 50% of his cases. Several factors influence the ability to detect immunoglobulin in B-cell tumors, and negative staining of paraffin-embedded tissues does not preclude a B-cell origin. Fixation and processing decreases the immunoreactivity of tissue antigens.19 It has been our experience that this is proportional to the time the tissue remains in fixative. Oral pathology laboratories often receive most of their specimens via the Postal Service, which usually results in a fixation time of 48 to 96 hours or longer. Moreover, because of the relatively low numbers of biopsies performed by most dental clinicians, the 10% buffered formalin fixative provided by the laboratory may degrade on the shelf over periods of months to years before use. Probably a higher percentage of immunoglobulincontaining tumors could have been detected if these tissues had been consistently fixed in fresh 10% buffered formalin for a maximum of 24 to 48 hours.
Handlers
366
Table
et al.
II. Summary
Oral April,
of immunoperoxidase
staining
results Case No. I
Histologic diagnosis (Lukes-Collins)
35
SCFC’? SCFCCN LCFCCD LCFCCN LCFCC?+ SNCFCC?’ SNCFCCD SNCFCCD SNCFCCD* SNCFCC” SNCFCCD SNCFCC” SNCFCC?* SNCFCC? LNCFCG’ LNCFCCD LNCFCCN LNCFCCD LNCFCC? LNCFCCD LNCFCC? LNCFCC? lBSD lBSD IBS lBSD P/MD P/MD P/MD P/MD P/MD P/MD Plasmacytoid HistiocyticD Inflammatory
36
Inflammatory
2 3 4 5 6 I 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 29 30 31 32 33 34
NNodular DDiffuse *Burkitt
Immunoperoxidase pattern Negative Negative Negative Negative Negative Negative Negative Negative IgM Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative K
W, K IgA, K I&, K IgM, K
lymphocyticD
L L IgM, K L I&, L Muramidase I&, IgM, Ig& Muramidase IgG, IgM, &A, Muramidase
K L K L
pattern (Rappaport) pattern (Rappaport). type.
Although we did not routinely include pretreatment of the tissue with trypsin to increase antigenicity, trypsinization probably facilitates demonstration of small amounts of intracellular immunoglobulin.20 These factors may account for the higher percentage of positively staining tumors reported by Matthews and Basu,3 in contrast to those of Taylor’ and ourselves. Some B-cell lymphomas do not produce immunoglobulin, thus precluding positive staining.’ In spite of these variables, all of the tumors classified as immunoblastic sarcoma, plasmacytoma, and plasmacytoid lymphocytic lymphoma stained positively. Tumors of these types are composed of
Surg. 1986
cells that represent the most advanced stages of B-cell transformation. These results are similar to the findings of Wright and associates5 in their study of formalin-fixed and paraffin-embedded oral plasma cell lesions in which four of four stained positively. This probably is related to the presence of greater amounts of cytoplasmic immunoglobulin in cells showing more advanced transformation, as was demonstrated in Taylor’s immunoperoxidase studies.*’ The knowledge that routinely fixed and processed samples of these types of malignant B-cell neoplasm stain positively for immunoglobulins can be of great value to the surgical pathologist in sorting through the diagnostic dilemmas that these types of tumor may present. Oral plasma cell tumors are often difficult to differentiate from inflammatory processes characteristically composed predominantly of plasma cells. The presence of a monoclonal staining pattern can serve to confirm the neoplastic process (Fig. 2). Immunoblastic sarcomas are often morphologically similar to amelanotic melanomas and undifferentiated carcinomas, and immunoperoxidase staining is useful in the differentiation of these tumors (Fig. 3). Only two of twenty-two cases of follicular centercell lymphoma showed positive staining. Both of these were transformed follicular center cell lymphomas (one SNCFCC and one LNCFCC), which should contain more cytoplasmic immunoglobulin than nontransformed follicular center cell lymphomas (SCFCC and LCFCC) but less cytoplasmic immunoglobulin than immunoblastic or plasma cell tumors. Transformed follicular center cell lymphomas might, therefore, be expected to stain positively more often than nontransformed follicular centercell tumors and less often than immunoblastic sarcoma or plasma cell tumors in paraffin-embedded tissues. Morphologic examination in correlation with immunoperoxidase staining is essential in establishing the diagnosis of follicular center cell lymphoma. Except for small-cell lymphocytic lymphoma, all other categories of B-cell lymphoma were represented in our series. Twenty-two of the thirty-three cases (67%) of B-cell lesions examined were follicular center cell lymphomas. This is comparable to a study of 250 nodal B-cell lymphomas by Taylor,14 in which 53% represented follicular center cell lymphomas. The majority of these extranodal oral lymphomas (77%) were transformed follicular center cell lymphomas (SNCFCC and LNCFCC) (Fig. l), which is a much higher percentage than that seen in lymphomas from a variety of locations studied by Vanderbilt University, where only 36% of the follic-
Volume Number
Extranodal oral lymphoma
61 4
ular center cell lymphomas were of the transformed variety.22 Of our nine cases in the SNCFCC group, two were of the Burkitt type. Ultimately, both (one by us and one by another laboratory) of these stained positively for IgM and IgM, kappa, respectively. This is in accord with other studies in which most Burkitt cell lymphomas have been shown to be B-cell lesions in which the most common markers are IgM and kappa.23 SUMMARY
Most (97%) of the oral lymphomas in this study were morphologically identified as B-cell proliferations, with the majority of these (67%) being composed of follicular center cells. A high proportion (77%) of the follicular center cell lymphomas were of the transformed (SNCFCC and LNCFCC) type. Immunoperoxidase staining of formalin-fixed and paraffin-embedded tissues was found to be of little help in evaluating follicular center cell lymphomas, with the exception of the Burkitt type of SNCFCC lymphomas. On the other hand, this technique was highly useful in establishing the monoclonal B-cell nature of all of the immunoblastic sarcomas and plasma cell neoplasms. The diagnosis of a single true histiocytic lymphoma was also confirmed by immunoperoxidase staining. We would like to thank Mrs. Mary Wile and Ms. Barbara Ray for their expert help in the immunoperoxidaseand routine staining of the specimens. REFERENCES I. Taylor CR: lmmunohistologic studies of lymphomas: new methodology yields new information and poses new problems. J Histochem Cytochem 27: 1189-l 191, 1979. 2. Banks PM: Diagnostic applications of an immunoperoxidase method in hematopathology. J Histochem Cytochem 27: 1192-l 194, 1979. 3. Matthews JB, Basu MK: Primary extranodal lymphoma of the oral cavity: an immunohistochemical study. Br J Oral Surg 21: 159-170, 1983. 4. Matthews JB, Basu MK: Plasma cell lesions within the oral tissues: immunoperoxidase staining of routinely fixed and processed tissue. ORAL SURG ORAL MED ORAL PATHOL 54: 414-423, 1982. 5. Wright BA, Wysocki GP, Bannerjee MD: Diagnostic use of immunoperoxidase techniques for plasma cell lesions of the jaws. ORAL SURG ORAL MED ORAL PATHOL 52: 615-622, 1981. 6. Lukes RJ: The immunologic approach to the pathology of malignant lymphomas. Am J Clin Pathol 72: 657-669, 1979.
367
7. Lukes RJ, Taylor CR, Parker JW, Lincoln TL, Pattengale PK, Tindle BH: A morphologic and immunologic surface marker study of 299 cases of non-Hodgkin’s lymphomas and related leukemias. Am J Path01 90: 461-485, 1978. 8. Lukes RJ, Parker JW, Taylor CR, Tindle BH, Cramer AD, Lincoln TL: Immunologic approach to non-Hodgkin’s lymphomas and related leukemias: analysis of the results of multiparameter studies of 425 cases. Semin Hematol 15: 322-351, 1978. AJ, Simon RM, Osborne K, Merrill J, Young RC, 9. Garvin Berard CW: An autopsy study of histologic progression in non-Hodgkin’s lymphomas: 192 cases from National Cancer Institute. Cancer 52: 393-398, 1983. H: Tumors of the hematopoietic system, Sect 111, 10. Rappaport Fast. 8, Washington, D.C., 1966, Armed Forces Institute of Pathology. Il. Nathwani BN, Kim H, Rappaport H, Solomon J, Fox M: Non-Hodgkin’s lymphomas: a clinicopathologic study comparing two classifications. Cancer 41: 303-325, 1978. 12. Nathwani BN: A critical analysis of the classification of non-Hodgkin’s lymphomas. Cancer 44: 347-384, 1979. 13. Handlers JP, Meirose RJ, Abrams AM, Taylor CR: Lmmunoperoxidase technique in diagnosis of oral pemphigus vulgaris: an alternative method to immunofluorescence. ORAL SURG ORAL MED ORAL PATHOL 54: 207-212, 1982. 14. Taylor CR: Results of multiparameter studies of B-cell lymphomas. Am J Clin Pathol 72: 687-698, 1979. 15. Lennert K, Stein H, Kaiserring E: Cytological and functional criteria for the classification of malignant lymphomata. Br J Cancer 31 (Supp.):29-43, 1975. 16. Lukes RJ, Collins RD: New approaches to the classification of the lymphomata. Br J Cancer 31 (Supp.):l-27, 1975. 17. Blok P, Van Delden L, van der Waal I: Non-Hodgkin’s lymphoma of the hard palate. ORAL SURG ORAL MED ORAL PATHOL
47:
445-452,
1979.
18. Hashimoto N, Kurihara K: Pathologic characteristics of oral lymphomas. J Oral Pathol 11: 214-227, 1982. 19. Warnke R: Alteration of immunoglobulin-bearing lymphoma cells by, fixation. J Histochem Cytochem 27: 1195-l 196, 1979. 20. isaacson P, Wright DH: Anomalous staining patterns in immunohistologic studies of malignant lymphoma. J Histothem Cytochem 27: 1197-l 199, 1979. 21. Taylor CR: Immunoperoxidase techniques: theoretical and practical aspects. Arch Pathol Lab Med 102: 1 13-121, 1978. 22. Oviatt DL, Cousar JB, Flexner JM, Kurtin PJ, Collins RD, Stein RS: Malignant lymphoma of follicular center cell origin in humans. IV. Small transformed (noncleaved) cell lymphoma of non-Burkitt’s type. Cancer 52: 1196-1201, 1983. 23. Mann RB, Jaffe ES, Braylan RC, Nanba K, Frank MM, Ziegler JL, Berard CW: Non-endemic Burkitt’s lymphoma: a B-cell tumor related to germinal centers. N Engl J Med 295: 685-691, 1976. Reprint requests to: Dr. Janice P. Handlers Department of Pathology School of Dentistry-MC 0641 University of Southern California Los Angeles, CA 90089-0641
OF SOUTHERN UNIVERSITY
CALIFORNIA
SCHOOL
OF DENTISTRY
AND
D.D.S., * Los Angeles, Cal&
FACULTY
OF DENTISTRY.
Thirty-four cases of oral lymphoma were classified by the Lukes-Collins system on the basis of morphology and immunoperoxidase staining. Ninety-seven percent of these were morphologically identified as B-cell neoplasms: 6% SCFCC, 9% LCFCC, 26% SNCFCC, 24% LNCFCC, 12% IBS, and 16% malignant plasma cell proliferations. Monoclonal immunoperoxidase staining for cytoplasmic immunoglobulin was positive in 41% of the cases overall, but 100% of the cases of immunoblastic sarcoma and malignant plasma cell lesions stained positively. (ORAL SURC. ORAL MED. ORAL PATHOL. 61:362-367, 19861
T he use of the immunoperoxidase nique as an aid in the diagnosis
staining techof lymphomas occurring within lymph nodes has been well established in the literature.‘** There are few reports, however, of its use in extranodal disease, particularly that found in the head and neck area. Matthews and Basu reported a series of eleven cases of nonHodgkin’s lymphoma’ and eight cases of plasma cell lesions4 arising within the oral cavity in which this technique was used. Eight of the cases of nonHodgkin’s lymphoma and five of the plasma cell lesions stained positively for monoclonal immunoglobulin. Wright and associate9 examined four oral plasma cell lesions by immunoperoxidase methods, and all of them showed monoclonal staining. These studies suggest that the immunoperoxidase method is valuable in differentiating neoplastic lymphoid processes from inflammatory conditions and from nonlymphoid neoplasms on the basis of monoclonal This
project
was
funded
by
University
School of Dentistry Biomedical 2S07RR05303-21 and the Department University. *Department of Pathology. University School of Dentistry. **Division Faculty
362
of Oral of Dentistry,
Pathology, Dalhousie
of Southern Research of Oral
Department University.
of
California
Support Grant Biology, Dalhousie Southern of
California Oral
Biology,
staining. In addition, the immunoperoxidase technique is of value in determining the lineage of the neoplastic population, particularly when correlated with the morphologic appearance of the cell.6 It has been shown that approximately 50% of B-cell lymphomas will retain antigenicity of immunoglobulin markers after routine formalin fixation and processing.’ On the basis of a large number of cases studied by multiple morphologic and immunologic parameters, Lukes and Collins have outlined morphologic characteristics which are even more reliable than immunohistochemical stains of processed tissue in differentiating B-cell and T-cell lymphomas.7~8 The characteristics of B-ceil lymphomas are summarized in Table I. The prognostic significance of the Lukes and Collins classification of lymphomas is still somewhat controversial. However, it appears that immunoblastic sarcomas and noncleaved follicular center-cell (transformed) lymphomas have a poorer prognosis.9 It is well documented that there is a significant difference in the prognosis for nodular and diffuse lymphomas as classified by Rappaport.” In nodal lymphomas, the nodular lymphomas appear to have a distinctly better survival rate than those with a diffuse pattern of infiltration.gx “2 ‘* In this study, forty-one cases of potentially malig-
Volume Number
Extranodal
61 4 TABLE
1:
LUKES-COLLINS
CLASSIFICATION
OF
B-CELL
NU&W
Cyt0plasmic
Characteristics
Characteristics
Characteristics
1. Small Lymphocytic Lymphoma
Small cell 6 - 9 urn Uniform
Clumped chromati” Round, distinct border Rare mitoses
scanty Indistinct
2.
Small Cleaved Follicular Center Ceil (SCFCC)
Small cell 6. 12 urn Slight variation
Marked Irregularities Indented, Lobulated, Cleaved Clumped, dense chromatin Few-rare mitoses
Indistinct
3.
Large Cleaved Follicular Center Cell ILCFCC)
Large cell 20 40 urn Marked variation in size and shape
Coarse chromatin clumping Vesicular nuclei Marked irregularities Few-rare mitoses
Indistinct
4.
Small Non-Cleaved Follicular Center Cell (SNCFCC)
Medium
Multiple distinct nucleoli Distinct nuclear borders Rounded Numerous mitoses
Indistinct
5.
Large Non-Cleaved Follicular Center Cell (LNCFCC)
Large cell 20 - 40 urn
Vesicular Small nucleoli on nuclear membrane Irregularity in size & shape Numerous mitoses
I “creased Deep staining Lacks plasmacytoir’ features
6.
lmmunoblastic Sarcoma (16s)
Large cell 20
One or mcwe prominent nucleoli Much variation in size Dense chromatin clumping on nuclear membrane Numerous mitoses
Moderate to abundant Eosinophilic Distinct membrane Marked pyroninophilia Plasmacytoid
7.
Plasmacytoid Lvmphocytic Lymphoma
Small cell 6
Small, round Uniform Plasmacytoid Few to no mitoses
Distinct Eosinophilic Eccentric nucleus Plahmacytoid
6.
Plasmacytoma/ Multiple Myeloma
Ovoid cell Large cell > 20 urn
Distinct membrane Rounded Clockface (Peripheral) chromatin clumping Few mitoses
Abundant Eorinophilic Pyrinophilic Distinct membrane Eccentric nucleus
20 urn
40 urn
10 urn
nant lymphoid lesions were evaluated by routine stains and by immunohistochemical means to determine the number of routinely fixed and processed oral cases in which the lymphocytic infiltrate stained in a monoclonal manner by the immunoperoxidase technique using polyclonal test sera. Two of these were eliminated from the study because they were interpreted to be Hodgkin’s lymphoma, and three were eliminated because they were interpreted and confirmed by other studies to be leukemic infiltrates. The tumors were also classified according to the method of Lukes and Collins in order to determine what percentages of these oral lymphomas were composed of the different cell types described in this classification system (B-cell, T-cell, histiocytic, etc.). In a subsequent article the results of the morphologic and immunoperoxidase study of oral lymphomas will be correlated with clinical features and prognosis.
METHODS
363
LYMPHOMAS
C.dlldJ~
cell 15
oral lymphoma
AND MATERIALS
All cases with diagnoses of malignant lymphoma, lymphoproliferative process, plasmacytoma, multiple myeloma, or malignant tumor suggestive of lymphoma or plasmacytoma, received between January 1974 and January 1984, were selected from the files of the University of Southern California Oral Pathology Laboratory. Clinically, these lesions were all extranodal, located within intraoral soft tissue, mandible, or maxilla. Paraffin-embedded tissue was available in forty-one cases. Hematoxylin and eosin, methyl green pyronine, periodic acid-Schiff, and Giemsa stains were performed in each case. Specimens were also stained by the immunoperoxidase technique for IgG (1:500), IgM (1:300), IgA (1:300) and IgD (most cases) (1:300), kappa (l:lOOO), lambda (l:lOOO), and muramidase (1:300) as described previously. l3 All materials were obtained
364
0”
Handlers et al.
SCFCC
.
LCFCC
SNCFCC
LNCFCC
Oral April,
IBS
P,M
Surg. 1986
Lym#locytic
1. Total numberof caseswithin eachLukes-Collins category plotted in order of advancingB-cell transformation.
Fig.
from DAK0 of Santa Barbara, California, and Hornby, Ontario, with the exception of the rabbit PAP reagent, which was obtained from Zymed of Hornby, Ontario. Briefly, paraffin sections were deparaffinized and rehydrated. They were then placed in a hydrogen peroxide-methanol bath for 30 minutes at room temperature to block endogenous peroxidase activity. After a 5-minute bath in Tris buffer, pH 7.6, they were treated with the appropriate rabbit anti-human antisera and incubated for 30 minutes. Another 5-minute Tris wash was followed by application of swine anti-rabbit immunoglobulin, a 20-minute incubation, and a 5-minute wash. Rabbit PAP reagent was applied and incubated for 20 minutes. A Tris buffer wash preceded application of the chromagen diaminobenzidine prior to incubation of the tissue for 5 minutes. The slides were then counterstained with Mayer’s hematoxylin, dehydrated, and mounted with Permount. Tonsillar tissue and inflamed gingival tissue were used for positive controls. Negative controls were achieved by omitting the primary antibody and substituting normal swine serum. All sections were then studied and classified by the Lukes and Collins system of classification as
described for nodal lymphomas.7*8 The immunoperoxidase sections were evaluated for positivity against the respective controls. These results are tabulated in Table II. RESULTS
Of the forty-one cases, five were eliminated from the study because they were interpreted morphologically to represent Hodgkin’s disease (two cases) or myelomonocytic leukemic infiltrates (three cases). Two of the cases showed polyclonal staining for IgG, IgA, IgM, kappa, lambda, and muramidase. These
Fig. 2. A, Malignant plasmacell tumor showingnegative immunoperoxidase staining for lambda light chains. (Magnification, x250.) B, Samecasedemonstratingpositive monoclonalintracytoplasmicstaining for kappa light chains. This is demonstratedby the presenceof dark granular material within the cytoplasm,in contrast to the clear cytoplasmseenin A. (Magnification, x250.)
were interpreted on this basis, as well as morphologically, to represent inflammatory lesions and were not considered further. Fourteen of the remaining thirty-four cases (41%) showed monoclonal cytoplasmic staining. Classification of the tumors by the Lukes and Collins classification revealed that 6% of the thirtyfour cases were small-cleaved follicular center cell (SCFCC) lymphomas, 9% were composed of largecleaved follicular center cells (LCFCC), 26% of small noncleaved (small transformed) follicular center cells (SNCFCC), and 24% of large noncleaved (large transformed) follicular center cells (LNCFCC). Immunoblastic sarcoma (IBS) accounted for 12% of the cases, malignant plasma cell proliferations (plasmacytoma or multiple myeloma) for 18%, and plasmacytoid lymphocytic lymphoma for 3%. One case (3%) of true (muramidase-positive) histiocytic lymphoma was found (Fig. 1). None of the small-cleaved or large-cleaved follicu-
Volume Number
Extranodal
61 4
oral lymphoma
365
% loo-
Fig. 3. Immunoblastic sarcoma stained with periodic acid-Schiff stain.The cellsshowprominentnucleoli,dense chromatin clumping on the nuclear membrane,moderate to abundant cytoplasm with a distinct membrane,and numerousmitoses.(Magnification, x250.)
lar center cell lymphomas stained for immunoglobulin or muramidase. Eleven percent (one of nine) of the small noncleaved follicular center cell lymphomas stained positively. This case was positive for IgM only and was one of two cases of SNCFCC lymphoma of the Burkitt type in our series. Interestingly, although the other case was negative in our series, a repeat biopsy studied at another laboratory was positive for IgM and kappa chain. One of eight (13%) of the large noncleaved follicular center cell lymphomas stained positively (K only). In contrast to the above-mentioned tumors, all tumors (100%) classified as plasmacytoma/multiple myeloma (six of six) (Fig. 2), immunoblastic sarcoma (four of four) (Fig. 3), and plasmacytoid lymphocytic lymphoma (one of one) stained positively (Fig. 4). DISCUSSION
Large multiparameter studies of nodal lymphomas have shown that B-cell neoplasms account for the majority of non-Hodgkin’s lymphoma.9, 14-16Studies of extranodal oral lymphomas, although few, also appear to support this contention. In a morphologic study of eight cases of non-Hodgkin’s lymphoma of the hard palate by Blok and associates,17 seven were interpreted as B-cell neoplasms. Hashimoto and Kurihara** interpreted all of their nine cases of oral lymphoma as B-cell neoplasms on the basis of morphologic appearance. The results of our study were consistent with these findings. On the basis of morphologic examination, all but one of the thirtyfour cases were classified as B-cell lymphomas. The one exception was classified as a “true” histiocytic
0
I SCFCC -B-CELL
1 1 SNCFCC 1 LCFCC LNCFCC TR*NSFORM*TION
I ISS
I PIM
1 Plaamacytoid Lymphocytic m
Fig. 4. Percentageof tumorswithin eachLukes-Collins category showingpositive monoclonalstaining for cytoplasmic immunoglobulinplotted in order of advancing B-cell transformation.
lymphoma, and this was supported by positive immunoperoxidase stains for muramidase. Of the remaining thirty-three cases with B-cell morphology, thirteen (39%) were confirmed on the basis of monoclonal immunoperoxidase stains for cytoplasmic immunoglobulin to represent B-cell neoplasms. Other similar studies have reported a higher percentage of lymphomas showing positive staining for immunoglobulins. Matthews and Basu,3 in a study of eleven oral lymphomas, reported positive staining in 64%. In studies of nodal lymphomas, Taylor’ has reported positive monoclonal patterns in approximately 50% of his cases. Several factors influence the ability to detect immunoglobulin in B-cell tumors, and negative staining of paraffin-embedded tissues does not preclude a B-cell origin. Fixation and processing decreases the immunoreactivity of tissue antigens.19 It has been our experience that this is proportional to the time the tissue remains in fixative. Oral pathology laboratories often receive most of their specimens via the Postal Service, which usually results in a fixation time of 48 to 96 hours or longer. Moreover, because of the relatively low numbers of biopsies performed by most dental clinicians, the 10% buffered formalin fixative provided by the laboratory may degrade on the shelf over periods of months to years before use. Probably a higher percentage of immunoglobulincontaining tumors could have been detected if these tissues had been consistently fixed in fresh 10% buffered formalin for a maximum of 24 to 48 hours.
Handlers
366
Table
et al.
II. Summary
Oral April,
of immunoperoxidase
staining
results Case No. I
Histologic diagnosis (Lukes-Collins)
35
SCFC’? SCFCCN LCFCCD LCFCCN LCFCC?+ SNCFCC?’ SNCFCCD SNCFCCD SNCFCCD* SNCFCC” SNCFCCD SNCFCC” SNCFCC?* SNCFCC? LNCFCG’ LNCFCCD LNCFCCN LNCFCCD LNCFCC? LNCFCCD LNCFCC? LNCFCC? lBSD lBSD IBS lBSD P/MD P/MD P/MD P/MD P/MD P/MD Plasmacytoid HistiocyticD Inflammatory
36
Inflammatory
2 3 4 5 6 I 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 29 30 31 32 33 34
NNodular DDiffuse *Burkitt
Immunoperoxidase pattern Negative Negative Negative Negative Negative Negative Negative Negative IgM Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative K
W, K IgA, K I&, K IgM, K
lymphocyticD
L L IgM, K L I&, L Muramidase I&, IgM, Ig& Muramidase IgG, IgM, &A, Muramidase
K L K L
pattern (Rappaport) pattern (Rappaport). type.
Although we did not routinely include pretreatment of the tissue with trypsin to increase antigenicity, trypsinization probably facilitates demonstration of small amounts of intracellular immunoglobulin.20 These factors may account for the higher percentage of positively staining tumors reported by Matthews and Basu,3 in contrast to those of Taylor’ and ourselves. Some B-cell lymphomas do not produce immunoglobulin, thus precluding positive staining.’ In spite of these variables, all of the tumors classified as immunoblastic sarcoma, plasmacytoma, and plasmacytoid lymphocytic lymphoma stained positively. Tumors of these types are composed of
Surg. 1986
cells that represent the most advanced stages of B-cell transformation. These results are similar to the findings of Wright and associates5 in their study of formalin-fixed and paraffin-embedded oral plasma cell lesions in which four of four stained positively. This probably is related to the presence of greater amounts of cytoplasmic immunoglobulin in cells showing more advanced transformation, as was demonstrated in Taylor’s immunoperoxidase studies.*’ The knowledge that routinely fixed and processed samples of these types of malignant B-cell neoplasm stain positively for immunoglobulins can be of great value to the surgical pathologist in sorting through the diagnostic dilemmas that these types of tumor may present. Oral plasma cell tumors are often difficult to differentiate from inflammatory processes characteristically composed predominantly of plasma cells. The presence of a monoclonal staining pattern can serve to confirm the neoplastic process (Fig. 2). Immunoblastic sarcomas are often morphologically similar to amelanotic melanomas and undifferentiated carcinomas, and immunoperoxidase staining is useful in the differentiation of these tumors (Fig. 3). Only two of twenty-two cases of follicular centercell lymphoma showed positive staining. Both of these were transformed follicular center cell lymphomas (one SNCFCC and one LNCFCC), which should contain more cytoplasmic immunoglobulin than nontransformed follicular center cell lymphomas (SCFCC and LCFCC) but less cytoplasmic immunoglobulin than immunoblastic or plasma cell tumors. Transformed follicular center cell lymphomas might, therefore, be expected to stain positively more often than nontransformed follicular centercell tumors and less often than immunoblastic sarcoma or plasma cell tumors in paraffin-embedded tissues. Morphologic examination in correlation with immunoperoxidase staining is essential in establishing the diagnosis of follicular center cell lymphoma. Except for small-cell lymphocytic lymphoma, all other categories of B-cell lymphoma were represented in our series. Twenty-two of the thirty-three cases (67%) of B-cell lesions examined were follicular center cell lymphomas. This is comparable to a study of 250 nodal B-cell lymphomas by Taylor,14 in which 53% represented follicular center cell lymphomas. The majority of these extranodal oral lymphomas (77%) were transformed follicular center cell lymphomas (SNCFCC and LNCFCC) (Fig. l), which is a much higher percentage than that seen in lymphomas from a variety of locations studied by Vanderbilt University, where only 36% of the follic-
Volume Number
Extranodal oral lymphoma
61 4
ular center cell lymphomas were of the transformed variety.22 Of our nine cases in the SNCFCC group, two were of the Burkitt type. Ultimately, both (one by us and one by another laboratory) of these stained positively for IgM and IgM, kappa, respectively. This is in accord with other studies in which most Burkitt cell lymphomas have been shown to be B-cell lesions in which the most common markers are IgM and kappa.23 SUMMARY
Most (97%) of the oral lymphomas in this study were morphologically identified as B-cell proliferations, with the majority of these (67%) being composed of follicular center cells. A high proportion (77%) of the follicular center cell lymphomas were of the transformed (SNCFCC and LNCFCC) type. Immunoperoxidase staining of formalin-fixed and paraffin-embedded tissues was found to be of little help in evaluating follicular center cell lymphomas, with the exception of the Burkitt type of SNCFCC lymphomas. On the other hand, this technique was highly useful in establishing the monoclonal B-cell nature of all of the immunoblastic sarcomas and plasma cell neoplasms. The diagnosis of a single true histiocytic lymphoma was also confirmed by immunoperoxidase staining. We would like to thank Mrs. Mary Wile and Ms. Barbara Ray for their expert help in the immunoperoxidaseand routine staining of the specimens. REFERENCES I. Taylor CR: lmmunohistologic studies of lymphomas: new methodology yields new information and poses new problems. J Histochem Cytochem 27: 1189-l 191, 1979. 2. Banks PM: Diagnostic applications of an immunoperoxidase method in hematopathology. J Histochem Cytochem 27: 1192-l 194, 1979. 3. Matthews JB, Basu MK: Primary extranodal lymphoma of the oral cavity: an immunohistochemical study. Br J Oral Surg 21: 159-170, 1983. 4. Matthews JB, Basu MK: Plasma cell lesions within the oral tissues: immunoperoxidase staining of routinely fixed and processed tissue. ORAL SURG ORAL MED ORAL PATHOL 54: 414-423, 1982. 5. Wright BA, Wysocki GP, Bannerjee MD: Diagnostic use of immunoperoxidase techniques for plasma cell lesions of the jaws. ORAL SURG ORAL MED ORAL PATHOL 52: 615-622, 1981. 6. Lukes RJ: The immunologic approach to the pathology of malignant lymphomas. Am J Clin Pathol 72: 657-669, 1979.
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7. Lukes RJ, Taylor CR, Parker JW, Lincoln TL, Pattengale PK, Tindle BH: A morphologic and immunologic surface marker study of 299 cases of non-Hodgkin’s lymphomas and related leukemias. Am J Path01 90: 461-485, 1978. 8. Lukes RJ, Parker JW, Taylor CR, Tindle BH, Cramer AD, Lincoln TL: Immunologic approach to non-Hodgkin’s lymphomas and related leukemias: analysis of the results of multiparameter studies of 425 cases. Semin Hematol 15: 322-351, 1978. AJ, Simon RM, Osborne K, Merrill J, Young RC, 9. Garvin Berard CW: An autopsy study of histologic progression in non-Hodgkin’s lymphomas: 192 cases from National Cancer Institute. Cancer 52: 393-398, 1983. H: Tumors of the hematopoietic system, Sect 111, 10. Rappaport Fast. 8, Washington, D.C., 1966, Armed Forces Institute of Pathology. Il. Nathwani BN, Kim H, Rappaport H, Solomon J, Fox M: Non-Hodgkin’s lymphomas: a clinicopathologic study comparing two classifications. Cancer 41: 303-325, 1978. 12. Nathwani BN: A critical analysis of the classification of non-Hodgkin’s lymphomas. Cancer 44: 347-384, 1979. 13. Handlers JP, Meirose RJ, Abrams AM, Taylor CR: Lmmunoperoxidase technique in diagnosis of oral pemphigus vulgaris: an alternative method to immunofluorescence. ORAL SURG ORAL MED ORAL PATHOL 54: 207-212, 1982. 14. Taylor CR: Results of multiparameter studies of B-cell lymphomas. Am J Clin Pathol 72: 687-698, 1979. 15. Lennert K, Stein H, Kaiserring E: Cytological and functional criteria for the classification of malignant lymphomata. Br J Cancer 31 (Supp.):29-43, 1975. 16. Lukes RJ, Collins RD: New approaches to the classification of the lymphomata. Br J Cancer 31 (Supp.):l-27, 1975. 17. Blok P, Van Delden L, van der Waal I: Non-Hodgkin’s lymphoma of the hard palate. ORAL SURG ORAL MED ORAL PATHOL
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18. Hashimoto N, Kurihara K: Pathologic characteristics of oral lymphomas. J Oral Pathol 11: 214-227, 1982. 19. Warnke R: Alteration of immunoglobulin-bearing lymphoma cells by, fixation. J Histochem Cytochem 27: 1195-l 196, 1979. 20. isaacson P, Wright DH: Anomalous staining patterns in immunohistologic studies of malignant lymphoma. J Histothem Cytochem 27: 1197-l 199, 1979. 21. Taylor CR: Immunoperoxidase techniques: theoretical and practical aspects. Arch Pathol Lab Med 102: 1 13-121, 1978. 22. Oviatt DL, Cousar JB, Flexner JM, Kurtin PJ, Collins RD, Stein RS: Malignant lymphoma of follicular center cell origin in humans. IV. Small transformed (noncleaved) cell lymphoma of non-Burkitt’s type. Cancer 52: 1196-1201, 1983. 23. Mann RB, Jaffe ES, Braylan RC, Nanba K, Frank MM, Ziegler JL, Berard CW: Non-endemic Burkitt’s lymphoma: a B-cell tumor related to germinal centers. N Engl J Med 295: 685-691, 1976. Reprint requests to: Dr. Janice P. Handlers Department of Pathology School of Dentistry-MC 0641 University of Southern California Los Angeles, CA 90089-0641